低pH插入肽修饰的载siRNA脂质体的制备与体外评价  

Preparation and in Vitro Evaluation of pH Low Insertion Peptide-Modified Liposomes Encapsulating siRNA

作　　者：游利江 梁飓 曹德英[2] 杜青[2] 杨少坤[2] 向柏[2] YOU Li-jiang;LIANG Ju;CAO De-ying;DU Qing;YANG Shao-kun;XIANG Bai(Department of Clinical Pharmacy,the Second Affiliated Hospital of University of South China,Hengyang 421001,China;School of Pharmacy,Hebei Medical University,Shijiazhuang 050017,China)

机构地区：[1]南华大学附属第二医院临床药学科,湖南衡阳421001 [2]河北医科大学药学院,石家庄050017

出　　处：《中国药学杂志》2020年第4期312-316,共5页Chinese Pharmaceutical Journal

基　　金：基金资助:国家自然科学基金项目资助(81773666,81973251);河北省自然科学基金项目资助(H2019206445,H2019206434);河北医科大学2018年大学生创新性实验计划项目资助(USIP2018248)。

摘　　要：目的核酸类(siRNA等)药物在疾病的治疗上具有高度特异性、安全和靶点多样性等独特优势。但其游离形式在体内容易被核酸酶(RNase)降解、半衰期短、转染效率低,这大大限制了其临床应用。本实验拟设计构建一种微酸环境敏感的脂质体载体,用于实现siRNA(小干扰RNA)的肿瘤组织水平、细胞水平甚至细胞器水平的高效定位递送。方法采用薄膜分散法,以二油酰基磷脂酰乙醇胺(DOPE)和胆甾醇半琥珀酸酯(CHEMS)为膜材制备空白脂质体;用两亲性材料SA-R8压缩siRNA,再与空白脂质体孵育制备载siRNA脂质体;将端基功能化的磷脂(DSPE-PEG2000-MAL)与低pH插入肽(pHLIP)反应连接,再与载siRNA脂质体孵育融合,构建低pH插入肽修饰的载siRNA脂质体;借助动态光散射原理,流式细胞术和激光共聚焦技术,表征脂质体的粒径及分布,监测细胞摄取、胞内转运与分布特征。结果制备的载siRNA脂质体平均粒径在150~190 nm内;在pH 6.5环境下siRNA的细胞摄取量显著高于pH 7.4环境;并且siRNA能很好地定位在细胞质。结论该载体显示出了较强的pH敏感性,在微酸环境下可以显著提高siRNA的肿瘤细胞摄取水平。OBJECTIVE Therapeutic nucleic acids(siRNA etc.)have unique advantages such as high specificity,safety,and target diversity in the treatment of diseases.However,the naked siRNAs are easily degraded by nucleases(RNase),have short half-lives and low transfection efficiency,which limit their therapeutic application.To construct a mildly acidic microenvironment sensitive liposomal nanocarrier for the efficient localization and delivery of siRNA(small interfering RNA)at the tissue,cell and even organelle levels.METHODS Blank liposomes were prepared by thin-film hydration using dioleoyl phosphatidylethanolamine(DOPE)and cholesteryl hemisuccinate(CHEMS).The siRNA was compressed with the amphiphilic material SA-R8 to obtain SA-R8/siRNA,which was then incubated with blank liposomes to prepare liposomes encapsulating siRNA.The terminally functionalized phospholipid(DSPE-PEG2000-MAL)was linked to the low pH insertion peptide(pHLIP).The product was then incubated with liposomes encapsulating siRNA to construct pHLIP-modified liposomes encapsulating siRNA.The particle size and distribution of liposomes were characterized by dynamic light scattering principle.The cellular uptake,intracellular transport and distribution were monitored by flow cytometry and confocal laser-scanning technique.RESULTS The RESULTS showed that the average size of the prepared liposomes encapsulating siRNA was between 150 and 190 nm.The cell uptake of siRNA at pH 6.5 was significantly higher than that of pH 7.4.And the siRNA internalized was well localized in the cytoplasm.CONCLUSION This carrier shows strong pH sensitivity and can significantly increase the cell uptake of siRNA in the tumor acidic environment.

关 键 词：SIRNA 低pH插入肽 脂质体 酸敏感 抗肿瘤 

分 类 号：R944[医药卫生—药剂学]